Characterization of the RNA binding properties of Ku protein.
Ku protein, a heterodimer of 70 and 83 kDa polypeptides, is the regulatory component of the DNA-dependent protein kinase (DNA-PK). Ku protein binds to DNA ends and is essential for DNA double-strand break repair and V(D)J recombination. Although there is some evidence that Ku protein also binds RNA, its RNA binding properties have not been systematically explored. In the present study, Ku-binding RNAs were identified using systematic evolution of ligands by exponential enrichment (SELEX) technology. These RNAs were assigned to three classes based on common sequence motifs. Most of the selected RNAs bound to Ku protein with a Kd < or = 2 nM, comparable to the affinity of DNA fragments for Ku protein under similar conditions. Many of the RNAs inhibited DNA-PK activity by competing with DNA for a common binding site in Ku protein. None of several RNAs that were tested activated DNA-PK in the absence of DNA. The identification of diverse RNAs that bind avidly to Ku protein is consistent with the idea that natural RNAs may serve as modulators of DNA-PK activity. Moreover, the RNAs identified in this study may have utility as tools for experimental manipulation of DNA double-strand break repair activity in cells and cell extracts.
Yoo
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Program in Gene Regulation, Medical College of Georgia, Augusta 30912, USA.
Dynan
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D034961:Antigens, Nuclear; D001667:Binding, Competitive / Q000235:genetics; D002457:Cell Extracts / Q000737:chemistry; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D000072200:Ku Autoantigen; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D017423:Sequence Analysis, RNA
D034961:Antigens, Nuclear; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D012313:RNA; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen